Much epigenetic information is thought to be encoded in the identity and localization of potentially heritable chemical modifications to histone protein that package the genome, to which modification-contingent binding partners bind, thereby transducing downstream functional consequences. The fundamental repeating unit of chromatin, the nucleosome core particle, is a two-fold symmetric octamer of histone proteins enshrouded by two superhelical turns of DNA. This architecture places the two copies of each core histone in defined positions each projecting unstructured tails from the core of the structure to that are subject to dense posttranslational modification. For a given modification, is there meaningful information encoded by having two distinct modifiable sites per fundamental repeating unit of chromatin? There are hints that variation at this level is highly regulated, yet little is known about this scale of chromatin modifications owing to lack of tools that can measure these properties. We have developed a breakthrough calibrated ChIP technology that permits us to query this level of nucleosome sub-structure detail for the first time. In Aim 1 we will directly quantify the symmetry of histone modifications within nucleosomes with our calibrated ChIP method, then probe the function of this newly measurable chromatin property. Given that the unit of recognition for binding partners entire nucleosome and flanking DNA, as opposed to merely the tails, precisely how variation at this level spatially manifests is likely tobe an important element of discrimination. To this end, we have recently developed biochemical evidence that nucleosomal binding partners discriminate as a function of mark-symmetry. We seek to understand the mechanistic properties of this unprecedented level of recognition, both in its biophysical details and its functional consequences for cells and organisms. In Aim 2 we will define the molecular nature of bivalent domains-the seeming apposition of canonically activating and repressive histone modifications decorating critical developmental genes in pluripotent cells-- using calibrated sequential ChIP experiments calibrated with an exhaustive set of internal standards. We will then examine their biogenesis and predictive power as barriers to differentiation. We expect that the results of this study will illuminate the general principlesof sub-nucleosomal mark recognition and function, forming a compelling argument that this relatively un-explored level of chromatin modification is important for genome management.